Histamine releaser contained in human sweat

ABSTRACT

Provided are a sweat allergy antigen, an antibody capable of binding to the antigen specifically, and others, which are produced utilizing a microorganism-originated protein that exists in sweat allergy patient in a dissolved state or a partial peptide of the protein.

A computer readable text file, entitled “SequenceListing.txt,” createdon or about Mar. 10, 2015 with a file size of about 15 kb contains thesequence listing for this application and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

This application claims priority to Japanese Patent Application No.2012-145814, filed Jun. 28, 2012 and incorporated by reference herein inits entirety.

The present invention relates to clinical application of a novelhistamine releaser contained in human sweat (such as measurement ofantigen-specific antibody concentration in a patient serum, preparationof an antibody to a sweat antigen, and a specific hyposensitizationtherapy).

BACKGROUND ART

Most of atopic dermatitis patients exhibit an immediate-type allergicreaction to their own sweat and a roughly purified sweat antigen. Aroughly purified sweat antigen and an antibody specifically bindingthereto have been reported (Patent Document 1 and Patent Document 2).However, a real molecule (sweat antigen) thereof is unknown.

CITATION LIST Patent Documents

-   Patent Document 1: WO 2005-005474-   Patent Document 2: WO 2009-133951

SUMMARY OF THE INVENTION

The present inventors have purified a human sweat antigen from humansweat using a histamine release activity from peripheral blood basophilsof atopic dermatitis patient as an index and have identified MGL_1304,which is a protein derived from Malassezia globosa, as the human sweatantigen by mass spectrometry.

In one aspect, the present invention provides a sweat allergy antigenprotein that is a protein derived from a microorganism. For example, thepresent invention provides a protein consisting of an amino acidsequence represented by SEQ ID NO: 1.

In one aspect, the present invention provides an MGL_1304 partialpeptide. For example, the present invention provides a peptideconsisting of an amino acid sequence represented by any one of SEQ IDNOs: 2 to 7.

In one aspect, the present invention provides a gene encoding a sweatallergy antigen protein that is a protein derived from a microorganismand a gene encoding a MGL_1304 partial peptide.

In one aspect, the present invention provides an antibody or an antibodyfragment specifically binding to a sweat allergy antigen protein that isa protein derived from a microorganism or a MGL_1304 partial peptide.

In one aspect, the present invention provides a composition or kit fordetecting a sweat allergy antigen, or a composition or kit for measuringan amount of a sweat allergy antigen, comprising an antibody or anantibody fragment specifically binding to a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide.

In one aspect, the present invention provides a composition or kit fordetecting an antibody binding to a sweat allergy antigen, or acomposition or kit for measuring an amount of a antigen binding to asweat allergy antigen, comprising a sweat allergy antigen protein thatis a protein derived from a microorganism or a MGL_1304 partial peptide.

In one aspect, the present invention provides a composition or kit fordiagnosing a sweat allergy or a disease related to a sweat allergyantigen, comprising:

(i) a sweat allergy antigen protein that is a protein derived from amicroorganism or a MGL_1304 partial peptide; or

(ii) an antibody or an antibody fragment specifically binding to thesweat allergy antigen protein that is a protein derived from amicroorganism or a MGL_1304 partial peptide.

In one aspect, the present invention provides a composition fortreatment of a sweat allergy or a disease relating to a sweat allergyantigen comprising a sweat allergy antigen protein that is a proteinderived from a microorganism or a MGL_1304 partial peptide.

In one aspect, the present invention provides a composition for removalor neutralization of a sweat allergy antigen or a material for removinga sweat allergy antigen comprising an antibody specifically binding to asweat allergy antigen protein that is a protein derived from amicroorganism or a MGL_1304 partial peptide.

In one aspect, the present invention provides a composition or kit fordetermining an effect of a hyposensitization therapy comprising a sweatallergy antigen protein that is a protein derived from a microorganismor a MGL_1304 partial peptide.

In one aspect, the present invention provides a method for manufacturinga sweat allergy antigen protein comprising a step of culturingMalassezia globosa at pH 7 to pH 9, and a step of purifying a sweatallergy antigen protein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows that an amino acid sequence identical to MGL_1304 wasdetected by mass spectrometry after conducting further purification ofpartially purified sweat antigens (hereinafter also referred to as QRX).

A recombinant protein of MGL_1304 was produced by E. coli. FIG. 2 showsCBB staining and Western blotting analysis of the recombinant proteinusing an atopic dermatitis patient serum and an anti-his tag antibody.

FIG. 3 shows a histamine release assay of atopic dermatitis patients(AD1, AD2, and AD3) and a healthy person (HC1) for the recombinantprotein of MGL_1304.

FIG. 4 shows that pretreatment of atopic dermatitis patient serum withone of QRX and MGL_1304 leads to neutralization of binding of antibodywith the other one, respectively.

FIG. 5 shows that when atopic dermatitis patient serum is pretreatedwith MGL_1304 in a reaction system where the atopic dermatitis patientserum is reacted with a rat mast cell line expressing a humanhigh-affinity IgE receptor to sensitize a cell (to provide sensitivityto an antigen), a reactivity (ability of histamine release) of thesensitized cell to QRX disappeared.

In FIG. 6, A shows a prepared N-terminal or C-terminal truncated proteinof MGL_1304.

In FIG. 6, B shows a binding ability of the protein truncated at theN-terminal or the C-terminal of MGL_1304 to atopic dermatitis patientserum IgE. In FIGS. 6, C and D show histamine release activity of theprotein truncated at the N-terminal or the C-terminal of MGL_1304. InFIG. 6, E shows binding of an MGL_1304 recombinant protein and a partialpeptide thereof to a Smith2 antibody, and serums (AD1 and AD2) derivedfrom two atopic dermatitis patients.

FIG. 7 shows that binding of MGL_1304 recombinant protein with IgEderived from serum of atopic dermatitis patients or healthy volunteerwas determined by ELISA, indicating that presence or absence of thebinding is consistent with a reactivity of basophils of the serum donorto purified sweat antigens.

FIG. 8 shows experimental results when an extract of Malassezia fungusbody (M.G. lysate), Malassezia culture supernatant (M.G. Sup), MGL_1304recombinant protein (rMGL) and a partially purified sweat antigen (QRX)were subjected to electrophoresis to perform immunoblotting using atopicdermatitis patient serum of one patient. In particular, FIG. 8 showsthat pretreatment of the serum with a fusion protein (rTF-MGL) ofTrigger Factor and rMGL resulted in disappearance of the bindingproperty of IgE to QRX and rMGL.

FIG. 9 shows experimental results when an extract of Malassezia fungusbody (M.G. lysate), Malassezia culture supernatant (M.G. Sup), MGL_1304recombinant protein (rMGL) and partially purified sweat antigen (QRX)were subjected to electrophoresis to perform the immunoblotting asperformed in FIG. 8 except for using an atopic dermatitis patient serumof one person different from the patient of the serum used in FIG. 8.

FIG. 10 shows experimental results when a purified extract of Malasseziafungus body (M. lysate (purified)), Malassezia culture supernatant (M.Sup (purified)) and a partially purified sweat antigen (sweat (QRX))were subjected to electrophoresis to perform immunoblotting using anatopic dermatitis patient serum of one patient.

In FIG. 11, A shows Western blotting results when an MGL_1304 gene or amite antigen gene fused with Myc-tag was expressed in COS7 cells and aculture supernatant thereof was electrophoresed to perform Westernblotting using an anti-Myc antibody.

In FIG. 11, B shows correlation of histamine releasability between apartially purified sweat antigen (QR) and the MGL_1304 recombinantprotein (rMGL_1304).

FIG. 12 shows sites of MGL_1304 protein recognized by monoclonalantibodies MGLab6-3, MGLab8-2, MGLab8-4, MGLab9-1, MGLab9-5, MGLab10-8,MGLab10-10, MGLab22-1, MGLab36-1 and MGLab40-1 created by immunizingmice with a recombinant MGL_1304 protein. ELISA was performed fortesting whether the antibodies recognize Trigger Factor (TF), a TF-fusedMGL_1304 protein (TF-MGL), a peptide (TF-P1) corresponding to an aminoacid sequence 1-50 of the TF-fused MGL_1304 protein, a peptide (TF-P2)corresponding to an amino acid sequence 46-100 of the TF-fused MGL_1304protein, a peptide (TF-P3) corresponding to an amino acid sequence96-140 of the TF-fused MGL_1304 protein, and a peptide (TF-P4)corresponding to an amino acid sequence 136-183 of the TF-fused MGL_1304protein. The results indicated that the monoclonal antibodies created byimmunizing mice with the recombinant MGL_1304 protein recognize theamino acid sequence 46-100 of the MGL_1304 protein except that MGLab6-3recognizes the amino acid sequence 136-183 of the MGL_1304 protein.

FIG. 13 shows detection of a sweat antigen-specific IgE antibody inserums of atopic dermatitis patients (AD1 and AD2) and a healthyvolunteer (Nor1). An ELISA plate was coated with a mouse monoclonalantibody 8-2 prepared by immunization with the MGL_1304 recombinantprotein as a primary antibody and QRX was allowed to bind thereto. TheIgE antibody binding to a complex of the mouse monoclonal antibody 8-2and QRX was detected from the serums.

FIG. 14 shows detection of the sweat antigen-specific IgE antibody inthe serums of the atopic dermatitis patients (AD1 and AD2) and thehealthy volunteer (Nor1). An ELISA plate was coated with the mousemonoclonal antibody 8-2 prepared by immunization with the MGL_1304recombinant protein as the primary antibody and rMGL was allowed to bindthereto. The IgE antibody binding to a complex of the mouse monoclonalantibody 8-2 and rMGL was detected from the serums.

FIG. 15 shows detection of the sweat antigen-specific IgE antibody inthe serums of the atopic dermatitis patients (AD1 and AD2) and thehealthy volunteer (Nor1). An ELISA plate was coated with the Smith2antibody used as the primary antibody and rMGL was allowed to bindthereto. The IgE antibody binding to a complex of the mouse monoclonalantibody 8-2 and rMGL was detected from the serums.

FIG. 16 shows detection of the sweat antigen-specific IgE antibody inthe serums of the atopic dermatitis patients (AD1 and AD2) and thehealthy volunteer (Nor1). An ELISA plate was coated with a mousemonoclonal antibody 6-3 prepared by immunization with the MGL_1304recombinant protein as the primary antibody and rMGL was allowed to bindthereto. The IgE antibody binding to a complex of the mouse monoclonalantibody 6-3 and rMGL was detected from the serums.

FIG. 17 shows detection of the sweat antigen-specific IgE antibody inthe serums of the atopic dermatitis patients (AD1 and AD2) and thehealthy volunteer (Nor1). An ELISA plate was coated with the mousemonoclonal antibody 8-2 prepared by immunization with the MGL_1304recombinant protein as the primary antibody and rMGL was allowed to bindthereto. The IgE antibody binding to a complex of the mouse monoclonalantibody 8-2 and rMGL was detected from the serums.

FIG. 18 shows detection of the sweat antigen-specific IgE antibody inserums of allergic rhinitis patients (allergic rhinitis 1-5), atopicdermatitis patients (AD1-8) and healthy volunteer (Nor1-3). An ELISAplate was coated with the MGL_1304 recombinant protein (rMGL) and abinding IgE antibody was detected from the serums. The serums werediluted 10 times (×10) and 20 times (×20) before being used in the test.The vertical axis indicates absorbance at 450 nm.

FIG. 19 shows detection of the sweat antigen-specific IgE antibody inthe serums of the allergic rhinitis patients (allergic rhinitis 1-5) andthe healthy volunteer (Nor1-3). An ELISA plate was coated with theMGL_1304 recombinant protein (rMGL) and a binding IgE antibody wasdetected from the serums. The serums were diluted 10 times (×10) beforebeing used in the test. The vertical axis indicates absorbance at 450nm.

FIG. 20 shows time course of amount of antibodies binding to rMGL inserum of a patient subjected to a hyposensitization therapy by repeatedsubcutaneous injections of QRX. The blood of the patient was collectedthree times (March 2011 (2011.3), December 2011 (2011.12), and March2012 (2012.3)) to prepare the serums, and an rMGL-coated ELISA plate wasused for measuring a change in amount of anti-MGL human IgE antibodiesin the serums.

FIG. 21 shows time course of amount of antibodies binding to rMGL inserum of the patient subjected to the hyposensitization therapy byrepeated subcutaneous injections of QRX. The blood of the patient wascollected three times (March 2011 (2011.3), December 2011 (2011.12), andMarch 2012 (2012.3)) to prepare the serums, and an rMGL-coated ELISAplate was used for measuring a change in amount of anti-MGL human IgGantibodies in the serums.

FIG. 22 shows time course of amount of antibodies binding to rMGL inserum of the patient subjected to the hyposensitization therapy byrepeated subcutaneous injections of QRX. The blood of the patient wascollected three times (March 2011 (2011.3), December 2011 (2011.12), andMarch 2012 (2012.3)) to prepare the serums, which were diluted 10 times(×10), 50 times (×50), and 100 times (×100), and an rMGL-coated ELISAplate was used for measuring a change in amount of anti-MGL human IgG4antibodies.

FIG. 23 shows concentration of the MGL_1304 protein (protein coded bythe MGL_1304 gene) produced in culture supernatant when Malasseziaglobosa was cultured in buffer A (PBS/HEPES/glucose) at pH 4, pH 6, orpH 8 for 2 to 60 minutes.

EMBODIMENT FOR CARRYING OUT THE INVENTION

1. Sweat Allergy Antigen Protein

In a first aspect, the present invention provides a sweat allergyantigen protein that is a protein derived from a microorganism.

As used herein, microorganisms may be Malassezia globosa, and Malasseziaglobosa may be Malassezia globosa (No. MYA-4612) purchasable from ATCC,for example. The protein derived from a microorganism may be a proteinproduced by Malassezia globosa. The protein produced by Malasseziaglobosa may be a protein secreted outside a fungus cell or may be aprotein present in a fungus cell.

Examples of the proteins secreted outside the Malassezia globosa funguscell include a protein coded by an MGL_1304 gene (e.g., a proteincomprising an amino acid sequence represented by SEQ ID NO: 1) and aprotein present in a culture supernatant of Malassezia globosa andbinding to serum derived from a sweat allergy patient and/or smith2antibodies (antibody produced by a hybridoma of Accession No. FERMBP-11111). The protein encoded by the MGL_1304 gene (e.g., a proteinconsisting of the amino acid sequence represented by SEQ ID NO: 1) maybe a protein expressed in an appropriate host such as E. coli, COS7cells and Malassezia globosa. The amino acid sequence represented by SEQID NO: 1 is as follows:

SEQ ID NO 1: MVSLNIFSAAFVASLASAVFAAPSALERRAAPDNTVWVTSVADHCLILPRHKMSVGDSESPGNMRSFCTKPYSSKQGQLASDFWTKAHFKKTDKYVQITGCINPNVQSTLLSNDEGGQYDSNGGEGGRGNPAGSVCLGYSSYVELVEPAGNRACIRCCYDPSDCDVSQDEAGCETVIPGKYDC.

Examples of a protein present in the Malassezia globosa fungus cellinclude a protein present in a fungus cell of Malassezia globosa andbinding to serum derived from a sweat allergy patient and/or smith2antibodies.

The protein present in a culture supernatant of Malassezia globosa andbinding to serum derived from a sweat allergy patient and/or smith2antibodies may be a protein having a molecular weight of about 17 kDameasured by SDS-PAGE. The protein present in a cell of Malasseziaglobosa and binding to serum derived from a sweat allergy patient and/orsmith2 antibodies may be a protein having a molecular weight of about 30kDa measured by SDS-PAGE. “About 17 kDa” and “about 30 kDa” describedabove are reasonably recognized by those skilled in the art as molecularweights measured by experiments (SDS-PAGE). For example, “about 17 kDa”and “about 30 kDa” described above may be molecular weights notexceeding ranges of “from 14 kDa to 20 kDa” and “from 27 kDa to 33 kDa”,respectively. The protein present in a culture supernatant or a funguscell of Malassezia globosa and binding to serum derived from a sweatallergy patient and/or smith2 antibodies may be a protein havinghistamine releasing activity for basophils derived from a sweat allergypatient and/or a protein which induces histamine release via IgE from acell expressing an IgE receptor.

As used herein, a sweat allergy antigen is an antigenic substance(hereinafter also referred to as a sweat antigen) which is contained inhuman sweat and which induces an allergic reaction to cause diseasessuch as atopic dermatitis and cholinergic urticaria, and a sweat allergyantigen protein is a sweat allergy antigen that is a protein. In oneembodiment, the sweat allergy antigen protein is dissolved in sweat.

As used herein, the sweat allergy antigen has histamine releaseactivity. Histamine release activity can be determined in accordancewith a known method (Koro, O. et al., J. Allergy Clin. Immunol., 103,663-670, 1999). For example, histamine release activity may bedetermined by contacting a sweat allergy antigen and an IgE antibodywith a cell expressing an IgE receptor on a cell surface and measuringan amount of histamine secreted from the cell. Examples of cellsexpressing IgE receptors on the cell surfaces include, basophils, mastcells (mastocytes) and a cell line artificially prepared by expressingIgE receptor gene which is able to release a chemical transmitter suchas histamine.

For example, when an amount of histamine is measured and an amount offree histamine is within a range of from 3 to 97% relative to the totalamount of histamine, it can be determined that the histamine releaseactivity is positive (Koro, O. et al., J. Allergy Clin. Immunol., 103,663-670, 1999).

“sweat allergy antigen protein that is a protein derived frommicroorganisms” provided by the present invention may be prepared bypurification from a secretion of a sweat gland using the histaminerelease activity as an index. For example, the sweat allergy antigenprotein is prepared by a method comprising a step of concentrating humansweat, a step of purification using anion-exchange columnchromatography, a step of purification using reverse-phase columnchromatography, and a step of purification using gel filtration columnchromatography. Therefore, in one embodiment, the present inventionprovides a sweat allergy antigen protein that is a protein derived froma microorganism and that is prepared by a method comprising a step ofconcentrating human sweat, a step of purification using anion-exchangecolumn chromatography, a step of purification using reverse-phase columnchromatography, and a step of purification using gel filtration columnchromatography. In one embodiment, the present invention provides amethod of manufacturing a sweat allergy antigen protein that is aprotein derived from a microorganism comprising a step of concentratinghuman sweat, a step of purification using anion-exchange columnchromatography, a step of purification using reverse-phase columnchromatography, and a step of purification using gel filtration columnchromatography.

“sweat allergy antigen protein that is a protein derived from amicroorganism” provided by the present invention may be prepared from aculture of Malassezia globosa. Condition and method for the culture maybe appropriately selected by those skilled in the art and those are notlimited. For example, “sweat allergy antigen protein that is a proteinderived from a microorganism” provided by the present invention may beprepared by culturing Malassezia globosa (No. MYA-4612) available fromATCC in a 2693 mDixon medium (2693 mDixon medium is prepared by mixingMalt Extract (36 g); Desiccated Oxbile (20 g); Tween 40 (10 ml); Peptone(6.0 g); Glycerol (2.0 ml); Oleic Acid (2.0 ml); and DI Water (1.0 L) toacquire a solution adjusted to pH 6 by using HC1 and autoclaving thesolution put into a suitable container at 121° C.) at 32° C. for fourdays, centrifuging the culture (at 2000 rpm for 10 minutes), andpurifying an acquired supernatant or lysate (cell lysate) of funguscells dissolved in a phosphate buffer solution (PBS) using binding tothe Smith2 antibody as an index.

Therefore, in one embodiment, the present invention provides a sweatallergy antigen protein that is a protein derived from a microorganismand that is prepared by a step of culturing Malassezia globosa and astep of purifying a culture supernatant or fungus cell lysate usingbinding to the Smith2 antibody as an index. In one embodiment, thepresent invention provides a method of manufacturing a sweat allergyantigen protein that is a protein derived from a microorganismcomprising a step of culturing Malassezia globosa and a step ofpurifying a culture supernatant or fungus cell lysate using binding tothe Smith2 antibody as an index.

The production of the MGL_1304 protein (protein encoded by the MGL_1304gene), i.e., the sweat allergy antigen protein from Malassezia globosa,is increased when Malassezia globosa is cultured at pH 8 as compared towhen Malassezia globosa is cultured at pH 4 or pH 6. Therefore,Malassezia globosa may be cultured at pH 7 to 10, preferably pH 7 to 9,more preferably pH 8.

The purification of the sweat allergy antigen protein from a culturesolution of Malassezia globosa is performed as a more simple operationwhen the culture solution contains no protein other than the sweatallergy antigen protein. Therefore, after Malassezia globosa is culturedat pH 4 to 6, preferably pH 4 to increase a fungus cell amount, aculture supernatant is discarded and Malassezia globosa is subsequentlycultured by using a buffer solution (e.g., PBS/HEPES/glucose buffersolution) of pH 7 to 10, preferably pH 7 to 9, more preferably pH 8, soas to produce a sweat allergy antigen protein, and the sweat allergyantigen protein may be purified from the buffer solution containing theobtained sweat allergy antigen protein. The purification may beperformed by column chromatography (e.g., ion-exchange columnchromatography, reverse-phase column chromatography, and gel filtrationcolumn chromatography).

The “sweat allergy antigen protein that is a protein derived from amicroorganism” provided by the present invention may be prepared byexpressing a protein encoded by the MGL_1304 gene (e.g., a proteincomprising the amino acid sequence represented by SEQ ID NO: 1) in asuitable host such as a microorganism and a cell (e.g., E. coli, COS7cells, and Malassezia globosa).

Therefore, in one embodiment, the present invention provides a sweatallergy antigen protein that is a protein derived from a microorganismand that is prepared by expressing a protein encoded by the MGL_1304gene (e.g., a protein comprising the amino acid sequence represented bySEQ ID NO: 1) in a suitable host such as a microorganism and a cell(e.g., E. coli, COS7 cells, and Malassezia globosa). In one embodiment,the present invention provides a method of manufacturing a sweat allergyantigen protein that is a protein derived from a microorganismcomprising a step of expressing a protein encoded by the MGL_1304 gene(e.g., a protein consisting of the amino acid sequence represented bySEQ ID NO: 1) in a suitable host such as a microorganism and a cell(e.g., E. coli, COS7 cells, and Malassezia globosa).

In one embodiment, the present invention provides an analog of theprotein encoded by the MGL_1304 gene (e.g., a protein consisting of theamino acid sequence represented by SEQ ID NO: 1). The analog is notparticularly limited as long as the analog can be prepared based on theprotein encoded by the MGL_1304 gene or the protein consisting of theamino acid sequence represented by SEQ ID NO: 1. Examples of the analogof the protein consisting of the amino acid sequence represented by SEQID NO: 1 include a protein consisting of an amino acid sequence having60, 70, 80, 90, or 95% or more identity to the amino acid sequencerepresented by SEQ ID NO: 1, and a protein consisting of an amino acidsequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acids deleted, substituted, or added in the aminoacid sequence represented by SEQ ID NO: 1.

Another example of the analog of the protein encoded by the MGL_1304gene (e.g., a protein consisting of the amino acid sequence representedby SEQ ID NO: 1) may be a protein encoded by the MGL_1304 gene (e.g., aprotein consisting of the amino acid sequence represented by SEQ IDNO: 1) to which a tag is added. As used herein, a tag means a portionadded to a protein or a polypeptide for purification, detection, etc. ofthe protein or polypeptide and is exemplified by histidine (His),glutathione-S-transferase (GST), a maltose-binding protein (MBP), myc, aFLAG tag, Trigger Factor (TF), etc. A polypeptide having a tag addedthereto is obtained by expressing the polypeptide by using an expressionvector, for example, pET30a (manufactured by Novagen) (for His tags),pGEX (manufactured by GE Healthcare Bio-Sciences Corp.) (for GST tags),and a pCold TF vector (manufactured by TAKARA BIO INC.), in a suitablehost such as a microorganism and a cell (e.g., E. coli, COS7 cells, andMalassezia globosa).

The analog of the protein encoded by the MGL_1304 gene (e.g., a proteinconsisting of the amino acid sequence represented by SEQ ID NO: 1) isable to bind to the IgE antibody present in a serum of a sweat allergypatient.

The analog of the protein encoded by the MGL_1304 gene (e.g., a proteinconsisting of the amino acid sequence represented by SEQ ID NO: 1) mayhave the histamine release activity.

The protein encoded by the MGL_1304 gene (e.g., a protein consisting ofthe amino acid sequence represented by SEQ ID NO: 1) and the analogthereof provided by the preset invention can be manufactured by aconventional genetic engineering method or a method used in peptidesynthesis. The manufactured proteins and the analogs thereof may be usedas the sweat allergy antigen. Therefore, in one embodiment, the presentinvention provides a use of a protein derived from a microorganism or ananalog thereof (e.g., a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1 and an analog thereof, and a protein presentin a culture supernatant of Malassezia globosa and binding to serumderived from a sweat allergy patient and/or smith2 antibodies) inmanufacturing of the sweat allergy antigen.

The protein or polypeptide provided by the present invention binds tothe IgE antibody and/or the IgG antibody binding to a sweat allergyantigen present in a serum of a sweat allergy patient, and therefore canbe used for detecting, quantifying, or removing IgE antibody and/or IgGantibody binding to the sweat allergy antigen present in a serum of asweat allergy patient. Thus, in one embodiment, the present inventionprovides use of a protein derived from a microorganism (e.g., theproteins encoded by the MGL_1304 gene (e.g., a protein consisting of theamino acid sequence represented by SEQ ID NO: 1) and the analogsthereof) in detection, quantification, neutralization, or removal of IgEantibody and/or IgG antibody binding to a sweat allergy antigen.

The protein or polypeptide provided by the present invention may be anisolated protein or polypeptide.

2. MGL_1304 Partial Peptide

In a second aspect, the present invention provides an MGL_1304 partialpeptide.

The MGL_1304 partial peptide may be a sweat allergy antigen. Therefore,in one embodiment, the present invention provides use of the MGL_1304partial peptide in manufacturing of a sweat allergy antigen.

As used herein, the MGL_1304 partial peptide may be a peptidecorresponding to a portion of a protein encoded by the MGL_1304 gene.The MGL_1304 partial peptide may be a peptide consisting of a portion ofthe amino acid sequence represented by SEQ ID NO: 1. Examples of theMGL_1304 partial peptide include a peptide corresponding to a residue32-173 of the polypeptide represented by SEQ ID NO: 1 (SEQ ID NO: 2), apeptide corresponding to a residue 32-183 of the polypeptide representedby SEQ ID NO: 1 (SEQ ID NO: 3), a peptide corresponding to a residue27-173 of the polypeptide represented by SEQ ID NO: 1 (SEQ ID NO: 4), apeptide corresponding to a residue 27-183 of the polypeptide representedby SEQ ID NO: 1 (SEQ ID NO: 5), a peptide corresponding to a residue22-173 of the polypeptide represented by SEQ ID NO: 1 (SEQ ID NO: 6) ora peptide corresponding to a residue 22-183 of the polypeptiderepresented by SEQ ID NO: 1 (SEQ ID NO: 7).

Therefore, in one embodiment, the present invention provides a peptidecomprising an amino acid sequence represented by any of SEQ ID NOs: 2 to7.

The MGL_1304 partial peptide may be a peptide binding to the IgEantibody present in a serum of a sweat allergy patient.

In one embodiment, the MGL_1304 partial peptide may release histaminevia IgE from a cell expressing the IgE receptor.

The MGL_1304 partial peptide provided by the present invention may be anisolated protein or polypeptide.

In one embodiment, the present invention provides an analog of theMGL_1304 partial peptide. The analog is not particularly limited as longas the analog can be prepared based on a peptide consisting of a portionof the amino acid sequence represented by SEQ ID NO: 1. Examples of theanalog of the peptide consisting of a portion of the amino acid sequencerepresented by SEQ ID NO: 1 include a peptide consisting of an aminoacid sequence having 60, 70, 80, 90, or 95% or more identity to an aminoacid sequence represented by any of SEQ ID NOs: 2 to 7, and apolypeptide consisting of an amino acid sequence with 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsdeleted, substituted, or added in an amino acid sequence represented byany of SEQ ID NOs: 2 to 7.

Another example of the analog of the peptide consisting of a portion ofthe amino acid sequence represented by SEQ ID NO: 1 may be a peptideconsisting of an amino acid sequence represented by any of SEQ ID NOs: 2to 7 to which a tag is added. As used herein, a tag means a portionadded to a polypeptide for purification, detection, etc. of thepolypeptide and is exemplified by histidine (His),glutathione-S-transferase (GST), a maltose-binding protein (MBP), myc, aFLAG tag, Trigger Factor (TF), etc. A polypeptide having a tag addedthereto is obtained by expressing the polypeptide by using an expressionvector, for example, pET30a (manufactured by Novagen) (for His tags),pGEX (manufactured by GE Healthcare Bio-Sciences Corp.) (for GST tags),and a pCold TF vector (manufactured by TAKARA BIO INC.), in a suitablehost cell such as a microorganism and a cell.

These analogs of the MGL_1304 partial peptide can be analogs of peptidesbinding to IgE antibody present in a serum of a sweat allergy patient.

The analogs of the MGL_1304 partial peptide may release histamine viaIgE from a cell expressing IgE receptor.

The analogs of the MGL_1304 partial peptide provided by the presentinvention may be an isolated protein or polypeptide.

The MGL_1304 partial peptide and the analog thereof provided by thepreset invention can be manufactured by a conventional geneticengineering method or a method used in peptide synthesis. For example,the MGL_1304 partial peptide and the analog thereof may be manufacturedby the following method:

(i) the MGL_1304 partial peptide provided by the preset invention isexpressed in E. coli or cells using an expression vector into which anucleotide encoding an amino sequence represented by any of SEQ ID NOs:2 to 7 is inserted; or

(ii) the MGL_1304 partial peptide provided by the preset invention isexpressed in E. coli or cells using an expression vector inserted intowhich a nucleotide encoding an amino sequence having 60, 70, 80, 90, or95% or more identity to an amino acid sequence represented by any of SEQID NOs: 2 to 7 is inserted; or(iii) the MGL_1304 partial peptide provided by the preset invention isexpressed in E. coli or cells using an expression vector into which anucleotide encoding an amino sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids deleted,substituted, or added in an amino acid sequence represented by any ofSEQ ID NOs: 2 to 7 is inserted.

The MGL_1304 partial peptide and the analog thereof provided by thepreset invention bind to IgE antibody and/or IgG antibody binding to asweat allergy antigen present in a serum of a sweat allergy patient andtherefore can be used for detecting, quantifying, neutralizing, orremoving IgE antibody and/or IgG antibody binding to a sweat allergyantigen present in a serum of a sweat allergy patient. Thus, in oneembodiment, the present invention provides use of the MGL_1304 partialpeptide and the analog thereof (e.g., the proteins consisting of anamino acid sequence represented by any of SEQ ID NOs: 2 to 7 and theanalogs thereof) in detection, quantification, neutralization, orremoval of IgE antibody and/or IgG antibody binding to a sweat allergyantigen.

3. Gene, Vector, and Transformant

In a third aspect, the present invention provides a gene encoding asweat allergy antigen protein that is a protein derived from amicroorganism, the MGL_1304 partial peptide, or an analog thereof.

In one embodiment, the present invention provides a polynucleotideencoding an amino acid sequence represented by any of SEQ ID NOs: 1 to7.

In one embodiment, the present invention provides a polynucleotideconsisting of a nucleotide sequence having 60, 70, 80, 90, or 95% ormore identity to a polynucleotide encoding an amino acid sequencerepresented by any of SEQ ID NOs: 1 to 7, and a polynucleotideconsisting of a nucleic acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 bases deleted, substituted, or added in apolynucleotide encoding an amino acid sequence represented by any of SEQID NOs: 1 to 7.

The polynucleotide provided by the present invention may be an isolatedpolynucleotide.

The polynucleotide provided by the present invention can be introducedinto a vector and expressed in a host cell as needed.

Therefore, in one embodiment, the present invention provides a vectorcontaining the polynucleotide provided by the present invention, and ahost cell (transformant) in which the polynucleotide provided by thepresent invention is introduced.

The polynucleotide, the vector, and the host cell provided by thepresent invention can be used in manufacturing of the protein or peptideconsisting of an amino sequence represented by any of any of SEQ ID NOs:2 to 7 or the analog thereof. Therefore, in one embodiment, the presentinvention provides use of the polynucleotide, the vector, and the hostcell provided by the present invention in manufacturing of a sweatallergy antigen.

4. Antibody

In a fourth aspect, the present invention provides an antibody or anantibody fragment specifically binding to a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide.

In one embodiment, the present invention provides an antibody or anantibody fragment specifically binding to a protein encoded by theMGL_1304 gene (e.g., a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1), a peptide consisting of an amino acidsequence represented by any of SEQ ID NOs: 2 to 7 and SEQ ID NOs: 10 to13, or an analog thereof. For example, an antibody or an antibodyfragment specifically binding to a protein encoded by the MGL_1304 gene(e.g., a protein consisting of the amino acid sequence represented bySEQ ID NO: 1) can be prepared by administering a protein or a peptideconsisting of an amino acid sequence represented by any of SEQ ID NOs: 1to 7 and 10 to 13 or an analog thereof expressed in E. coli, COS7 cells,or Malassezia globosa as an antigen to a mammal (e.g., a mouse or arabbit).

In one embodiment, the present invention provides an antibody or anantibody fragment specifically binding to a protein present in a culturesupernatant of Malassezia globosa and binding to serum derived from asweat allergy patient and/or smith2 antibodies. For example, an antibodyor an antibody fragment specifically binding to a protein present in aculture supernatant of Malassezia globosa and binding to serum derivedfrom a sweat allergy patient and/or smith2 antibodies can be prepared byadministering a protein present in a culture supernatant of Malasseziaglobosa and binding to serum derived from a sweat allergy patient and/orsmith2 antibodies to a mammal (e.g., a mouse or a rabbit) as an antigen.

In one embodiment, the present invention provides a method ofmanufacturing an antibody or an antibody fragment specifically bindingto a protein encoded by the MGL_1304 gene (e.g., a protein consisting ofthe amino acid sequence represented by SEQ ID NO: 1) comprisingadministering a protein or a peptide consisting of an amino acidsequence represented by any of SEQ ID NOs: 1 to 7 and 10 to 13 or ananalog thereof, or a protein present in a culture supernatant ofMalassezia globosa and binding to serum derived from a sweat allergypatient and/or smith2 antibodies, to a mammal (e.g., a mouse or arabbit).

As used herein, antibodies include polyclonal antibodies and monoclonalantibodies.

As used herein, monoclonal antibodies include genetic recombinantmonoclonal antibodies artificially modified for the purpose of reducingheterologous antigenicity to a human, for example, chimera monoclonalantibodies, humanized monoclonal antibodies and human monoclonalantibodies.

A fragment of an antibody is a portion of an antibody specificallybinding to an antigen. A fragment of an antibody may be Fab (fragment ofantigen binding), F(ab′)2, Fab′, a single-chain antibody (single chainFv; hereinafter referred to as scFv), a disulfide stabilized antibody(disulfide stabilized Fv; hereinafter referred to as dsFv), a dimerizedV-region fragment (hereinafter referred to as a diabody), and a peptidecomprising CDR (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5,pp. 441-456, 1996). The antibodies and the fragments of antibodies canbe prepared by a well-known method in the industry (see, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1988; http://www.gene.mie-u.ac.jp/Protocol/Original/Antibody.html; U.S.Pat. Nos. 6,331,415, 5,693,761, 5,225,539, 5,981,175, 5,612,205,5,814,318, 5,545,806, 7,145,056, 6,492,160, 5,871,907, and 5,733,743).

The antibodies provided by the present invention may be used as a toolrecognizing a protein consisting of the amino acid sequence representedby SEQ ID NO: 1 present in a sample derived from a human, for example,human sweat or skin, to analyze an amount, distribution, a function,etc. of the protein.

The antibodies provided by the present invention may have a label boundthereto. Examples of the label include an enzyme, a fluorescentsubstance, a radioisotope, biotin, etc.

Examples of the enzyme include alkaline phosphatase, peroxidase, glucoseoxidase, tyrosinase, acid phosphatase, etc.

Examples of the fluorescent substance include fluorescein isothiocyanate(FITC), GFP, luciferin, etc.

Examples of the radioisotope include ¹²⁵I, ¹⁴C, ³²P, etc.

Examples of an antibody specifically binding to a sweat allergy antigenprotein, i.e., a protein derived from a microorganism, provided by thepresent invention include, but not limited to, the following (i) to(iii):

(i) an antibody produced by the hybridoma (Mouse-Mouse hybridomasmith-1) of Accession No. FERM BP-11110 (transferred from FERM P-21439)deposited to the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology on Apr.1, 2009;(ii) an antibody produced by the hybridoma (Mouse-Mouse hybridomasmith-2) of Accession No. FERM BP-11111 (transferred from FERM P-21440)deposited to the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology on Apr.1, 2009; and(iii) an antibody produced by the hybridoma (Mouse-Mouse hybridomasmith-8) of Accession No. FERM BP-11112 (transferred from FERM P-21697)deposited to the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology on Apr.1, 2009.5. Composition or Kit for Detecting or Quantifying Sweat Allergy Antigen

In a fifth aspect, the present invention provides a composition or kitfor detecting a sweat allergy antigen, or a composition or kit formeasuring an amount of a sweat allergy antigen, comprising an antibodyor an antibody fragment specifically binding to a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide.

The detection of a sweat allergy antigen and the measurement of anamount of a sweat allergy antigen are measured by any method. Forexample, the detection of sweat allergy antigens and the measurement ofan amount of a sweat allergy antigen can be performed using Westernblotting or ELISA.

Therefore, in one embodiment, the composition for detecting a sweatallergy antigen or the composition for measuring an amount of a sweatallergy antigen may be a composition comprising an antibody or anantibody fragment specifically binding to a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide for use in Western blotting or ELISA.

In one embodiment, the kit for detecting a sweat allergy antigen or thekit for measuring an amount of a sweat allergy antigen may be a kitcomprising a reagent necessary for Western blotting or ELISA. Examplesof the reagents necessary for Western blotting include an SDS-PAGE gel,a nitrocellulose membrane or PVDF membrane, the antibody provided in thefourth aspect of the present invention, a blocking solution (e.g., a BSAsolution, a milk protein solution), wash solution (phosphate buffercontaining a surfactant (e.g., PBS containing Tween20)), a luminescencedetection reagent, etc. Examples of the reagents necessary for ELISAinclude a plate (e.g., a 96-well plate), the antibody provided in thefourth aspect of the present invention, a blocking solution (e.g., a BSAsolution, a milk protein solution), wash solution (phosphate buffercontaining a surfactant (e.g., PBS containing Tween20)), a chromogenicsubstrate (e.g., TMB), etc.

The kit for detecting a sweat allergy antigen or the kit for measuringan amount of a sweat allergy antigen may include as a standard a proteinencoded by the MGL_1304 gene (e.g., a protein consisting of the aminoacid sequence represented by SEQ ID NO: 1) or a protein present in aculture supernatant of Malassezia globosa and binding to serum derivedfrom a sweat allergy patient and/or smith2 antibodies. Using a solutionat a known concentration of a protein encoded by the MGL_1304 gene(e.g., a protein consisting of the amino acid sequence represented bySEQ ID NO: 1) or a protein present in a culture supernatant ofMalassezia globosa and binding to serum derived from a sweat allergypatient and/or smith2 antibodies, an amount of a sweat allergy antigencan accurately be quantified. For example, using a plurality ofsolutions at known concentrations of a protein present in a culturesupernatant of Malassezia globosa and binding to serum derived from asweat allergy patient and/or smith2 antibodies, an amount of a sweatallergy antigen can be accurately quantified. The protein encoded by theMGL_1304 gene (e.g., a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1) may be prepared by expressing the geneprovided in the third aspect of the present invention in a host such asE. coli and COS7 cells.

Western blotting and ELISA can be conducted as needed by those skilledin the art.

For example, in the case of Western blotting, a sample containing asweat allergy antigen is electrophoresed using the SDS-PAGE gel; theelectrophoresed sample is transferred to the PVDF membrane; the membraneis reacted with an antibody against the sweat allergy antigen, i.e., themouse IgG antibody provided in the fourth aspect of the presentinvention, followed by an enzyme-labeled anti-mouse IgG antibody; and,subsequently, the sweat allergy antigen can be detected or quantifiedthrough the enzyme activity.

For example, in the case of ELISA, a sample containing a sweat allergyantigen is coated on a plate by adding the sample to the plate; thecoated sample is reacted with an antibody against the sweat allergyantigens, i.e., the mouse IgG antibody provided in the fourth aspect ofthe present invention, followed by an enzyme-labeled anti-mouse IgGantibody; and, subsequently, the sweat allergy antigen can be detectedor quantified through the enzyme activity. In another example, a sweatallergy antigen may be detected or quantified by sandwich ELISA wheretwo types of the antibodies against the sweat allergy antigen providedin the fourth aspect of the present invention are used. Among thesereactions, the reaction with the enzyme-labeled anti-mouse IgG antibodymay be omitted when the mouse IgG antibody provided in the fourth aspectof the present invention, which is enzymatically labeled, is used.

In one embodiment, the present invention provides a method of detectinga sweat allergy antigen or a method of measuring an amount of a sweatallergy antigen comprising subjecting the antibody provided in thefourth aspect of the present invention to reacting with a sample derivedfrom a human. Examples of a sample derived from a human include, but notlimited to, a human sweat, human skin washings, a solution of an extractof human skin, a human serum, a human plasma, etc.

In one embodiment, the present invention provides use of an antibody oran antibody fragment specifically binding to a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide provided in the fourth aspect of the present invention,for manufacturing a composition or kit for detecting a sweat allergyantigen, or a composition or kit for measuring an amount of a sweatallergy antigen.

6. Composition or Kit for Detecting or Quantifying Antibody Binding toSweat Allergy Antigen

In a sixth aspect, the present invention provides a composition or kitfor detecting an antibody binding to a sweat allergy antigen, or acomposition or kit for measuring an amount of an antibody binding to asweat allergy antigen, comprising a sweat allergy antigen protein thatis a protein derived from a microorganism or the MGL_1304 partialpeptide. Examples of the detected or quantified antibodies may be, butnot limited to, human IgE antibodies or human IgG antibodies (e.g., allthe human IgG antibodies binding to sweat allergy antigens, or humanIgG4 antibodies binding to sweat allergy antigens).

The detection of an antibody binding to a sweat allergy antigen and themeasurement of an amount of an antibody binding to sweat allergyantigens can be measured by any method. For example, the detection of anantibody binding to a sweat allergy antigen and the measurement of anamount of an antibody binding to a sweat allergy antigen can beperformed using Western blotting or ELISA.

Therefore, in one embodiment, the composition for detecting an antibodybinding to a sweat allergy antigen or the composition for measuring anamount of an antibody binding to a sweat allergy antigen may be acomposition comprising the sweat allergy antigen protein that is aprotein derived from a microorganism or the MGL_1304 partial peptide foruse in Western blotting or ELISA.

In one embodiment, the kit for detecting an antibody binding to a sweatallergy antigen or the kit for measuring an amount of an antibodybinding to a sweat allergy antigen may be a kit comprising a reagentnecessary for Western blotting or ELISA. Examples of the reagentsnecessary for Western blotting include an SDS-PAGE gel, a nitrocellulosemembrane or PVDF membrane, the sweat allergy antigen provided in thefirst aspect of the present invention (e.g., a protein encoded by theMGL_1304 gene (e.g., a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1), a protein present in a culture supernatantof Malassezia globosa and binding to serum derived from a sweat allergypatient and/or smith2 antibodies) or the MGL_1304 partial peptideprovided in the second aspect of the present invention (e.g., a peptideindicated by any of SEQ ID NOs: 2 to 7), a blocking solution (e.g., aBSA solution, a milk protein solution), wash solution (phosphate buffersolution containing a surfactant (e.g., PBS containing Tween20)), aluminescence detection reagent, etc. Examples of the reagents necessaryfor ELISA include a plate (e.g., a 96-well plate), the sweat allergyantigen provided in the first aspect of the present invention (e.g., aprotein coded by the MGL_1304 gene (e.g., a protein consisting of theamino acid sequence represented by SEQ ID NO: 1), a protein present in aculture supernatant of Malassezia globosa and binding to serum derivedfrom a sweat allergy patient and/or smith2 antibodies (Accession No.FERM BP-11111)) or the MGL_1304 partial peptide provided in the secondaspect of the present invention (e.g., a peptide indicated by any of SEQID NOs: 2 to 7), a blocking solution (e.g., a BSA solution, a milkprotein solution), wash solution (phosphate buffer solution containing asurfactant (e.g., PBS containing Tween20)), etc.

The kit for detecting an antibody binding to a sweat allergy antigen orthe kit for measuring an amount of an antibody binding to a sweatallergy antigen may comprise as a standard a protein encoded by theMGL_1304 gene (e.g., a protein consisting of the amino acid sequencerepresented by SEQ ID NO: 1) or a protein present in a culturesupernatant of Malassezia globosa and binding to serum derived from asweat allergy patient and/or smith2 antibodies. Using a solution at aknown concentration of these proteins, an antibody binding to theprotein can accurately be quantified. For example, using a plurality ofsolutions at known concentrations of a protein present in a culturesupernatant of Malassezia globosa and binding to serum derived from asweat allergy patient and/or smith2 antibodies, the antibody binding toa sweat allergy antigen can accurately be quantified based on an amountof the antigen.

Western blotting and ELISA can be conducted as needed by those skilledin the art.

For example, in the case of ELISA, the sweat allergy antigen proteinprovided in the first aspect of the present invention or the MGL_1304partial peptide provided in the second aspect of the present inventionis coated; a sample containing an antibody binding to a sweat allergyantigen is added thereto; the sample is reacted with an enzyme-labeledanti-human antibody (e.g., enzyme-labeled anti-human IgG antibody,enzyme-labeled anti-human IgM antibody, enzyme-labeled anti-human IgG4antibody); and the enzyme activity can be measured to detect or quantifythe sweat allergy antigen. In another example, the antibody against asweat allergy antigen provided in the fourth aspect of the presentinvention is coated; the antibody is reacted with the sweat allergyantigen protein provided in the first aspect of the present invention orthe MGL_1304 partial peptide provided in the second aspect of thepresent invention; a sample containing an antibody binding to a sweatallergy antigen is added thereto; the sample is reacted withenzyme-labeled anti-human antibody (e.g., enzyme-labeled anti-human IgGantibody, enzyme-labeled anti-human IgM antibody, enzyme-labeledanti-human IgG1 antibody); and the enzyme activity may be measured todetect or quantify the sweat allergy antigen.

In one embodiment, the present invention provides a method of detectingan antibody binding to a sweat allergy antigen or a method of measuringan amount of an antibody binding to a sweat allergy antigen comprisingsubjecting the sweat allergy antigen provided in the first aspect of thepresent invention or the MGL_1304 partial peptide provided in the secondaspect of the present invention to reacting with a sample derived from ahuman. The method may be performed in vitro. Examples of the samplederived from a human include, but not limited to, a human sweat, humanskin washings, a solution of an extract of human skin, a human serum, ahuman plasma, etc.

In one embodiment, the present invention provides use of the sweatallergy antigen protein provided in the first aspect of the presentinvention or the MGL_1304 partial peptide provided in the second aspectof the present invention, for manufacturing a composition or kit fordetecting an antibody binding to a sweat allergy antigen, or acomposition or kit for measuring an amount of an antibody binding to asweat allergy antigen.

7. Composition or Kit for Diagnosing Sweat Allergy or Disease Related toSweat Allergy Antigen

In a seventh aspect, the present invention provides a composition or kitfor diagnosing a sweat allergy or a disease related to a sweat allergyantigen, comprising:

(i) the sweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide; or

(ii) an antibody or an antibody fragment specifically binding to thesweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide.

As used herein, a disease related to a sweat allergy antigen may be adisease accompanied with a sweat allergy induced by an antigenicsubstance contained in sweat and may be, for example, atopic dermatitis,urticaria (e.g., cholinergic urticaria), allergic rhinitis, etc.

The composition or kit for diagnosing a sweat allergy or a diseaserelated to a sweat allergy antigen provided in the seventh aspect of thepresent invention can diagnose, or assist the diagnosis of, a sweatallergy or a disease related to a sweat allergy antigen throughdetection or quantification of the antibody binding to a sweat allergyantigen or the sweat allergy antigen.

In one embodiment, the composition or kit for detecting or quantifyingan antibody binding to a sweat allergy antigen provided in the sixthaspect of the present invention is available for the composition or kit,relating to the seventh aspect of the present invention, for diagnosinga sweat allergy or a disease relating to a sweat allergy antigencomprising (i) the sweat allergy antigen protein that is a proteinderived from a microorganism or the MGL_1304 partial peptide. Forexample, an amount of an antibody (e.g., IgE and/or IgG (e.g., all theIgGs and/or IgG4)) binding to a sweat allergy antigen in a sample (e.g.,serum or plasma) derived from blood of a human subject is measured byusing the kit provided in the sixth aspect of the present invention, andis compared with an amount of an antibody binding to a sweat allergyantigen in a sample derived from blood of a healthy person, and if theamount of an antibody binding to the sweat allergy antigens in thesample derived from blood of the subject is larger than the amount of anantibody binding to the sweat allergy antigen in the sample derived fromblood of a healthy person, the subject can be diagnosed with a sweatallergy or a disease related to a sweat allergy antigen, or having arisk of a sweat allergy or a disease related to a sweat allergy antigen.Alternatively, this measurement result of an amount of an antibodybinding to the sweat allergy antigen can be used for assisting adiagnosis of a sweat allergy or a disease relating to a sweat allergyantigen from clinical finding.

In one embodiment, the composition or kit for detecting a sweat allergyantigen, or the composition or kit for measuring an amount of a sweatallergy antigen, provided in the fifth aspect of the present inventionis available for the composition or kit, relating to the seventh aspectof the present invention, for diagnosing a sweat allergy or a diseaserelated to a sweat allergy antigen comprising (ii) an antibody or anantibody fragment specifically binding to the sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide. For example, an amount of a sweat allergy antigen in asample of sweat of a human subject is measured by the kit provided inthe fifth aspect of the present invention, and is compared with anamount of a sweat allergy antigen in a sample of sweat of a healthyperson, and if the amount of a sweat allergy antigen in the sweat of thesubject is larger than the amount of an sweat allergy antigen in thesweat of a healthy person, the subject can be diagnosed with a sweatallergy or a disease related to a sweat allergy antigen, or having arisk of a sweat allergy or a disease related to a sweat allergy antigen.Alternatively, this measurement result of an amount of a sweat allergyantigen can be used for assisting a diagnosis of a sweat allergy or adisease relating to a sweat allergy antigen from clinical finding.

The kit for diagnosing a sweat allergy or a disease relating to a sweatallergy antigen provided in the seventh aspect of the present inventionmay comprise as a standard a protein encoded by the MGL_1304 gene (e.g.,a protein consisting of the amino acid sequence represented by SEQ IDNO: 1) or a protein present in a culture supernatant of Malasseziaglobosa and binding to serum derived from a sweat allergy patient and/orsmith2 antibodies. Use of this standard enables accurate quantificationand enables an accurate diagnosis.

In one embodiment, the present invention provides a kit or compositionfor a skin test comprising (i) a sweat allergy antigen protein that is aprotein derived from a microorganism or the MGL_1304 partial peptide.The skin test may be, for example, a patch test, a scratch test, a pricktest, or an intradermal test. The skin test may enable to diagnose asubject with a sweat allergy or a disease relating to a sweat allergy orhaving a risk thereof, or the skin test may provide data for assistingthe diagnosis.

The kit for a skin test may further comprise a saline used as acomparative control.

The patch test is widely performed as a simple method of examination fora contact allergy in the dermatological field. Since the presence of acontact allergy leads to sensitization of not only a portion ofdermatitis but also the whole body skin, a cause of contact dermatitiscan be determined by artificially reproducing allergic contactdermatitis on a healthy skin. For example, the sweat allergy antigenprotein or the MGL_1304 partial peptide (e.g., a protein consisting ofan amino acid sequence represented by any of SEQ ID NOs: 1 to 7)provided in the first or second aspect of the present invention isdripped or applied to an adhesive tape, and the adhesive tape is affixedto the back, the upper arm, the thigh, etc. The determination is madeafter two days, three days, and one weak in accordance with the ICDRG(International Contact Dermatitis Research Group) scale. Thedetermination is indicated by (−) when no reaction exists, (+) when areagent portion of the adhesive tape is associated with erythema andedema, or (++) or (+++) depending on a degree of the reaction.

The scratch test and the prick test are examinations performed bydropping an antigen solution onto a skin surface, making a scratch onthe skin with a needle tip such that bleeding does not occur, andexamining a reaction after 15 to 20 minutes. If the antigen-specific IgEantibody exists on a surface of a mast cell in an upper layer of thedermis, the antibody reacts with the dropped antigen, and histamine andchemical transmitters in the mast cell are released, resulting in a redswollen spot. For example, the sweat allergy antigen protein or theMGL_1304 partial peptide (e.g., a protein consisting of an amino acidsequence represented by any of SEQ ID NOs: 1 to 7) provided in the firstor second aspect of the present invention can be used as an antigen forthe scratch test and the prick test.

The test for intracutaneous reactivity is a test performed by injectingan extremely small amount of antigen solution inside a thin skin andobserving whether the injected portion becomes red and swollen after acertain time so as to determine the presence of an allergy. For example,the sweat allergy antigen protein or the MGL_1304 partial peptide (e.g.,a protein consisting of an amino acid sequence represented by any of SEQID NOs: 1 to 7) provided in the first or second aspect of the presentinvention can be used as the antigen for the intradermal test.

In another embodiment, the present invention provides a kit orcomposition for a histamine release test comprising (i) the sweatallergy antigen protein that is a protein derived from a microorganismor the MGL_1304 partial peptide. In the histamine release test, a cellreaction is used to measure an amount histamine release from blood cells(basophils) due to antigen stimulation. For example, an amount ofhistamine released from blood cells (basophils) due to antigenstimulation may allow a diagnosis of a sweat allergy or a diseaserelating to a sweat allergy or having a risk thereof, or may providedata assisting the diagnosis.

The kit for a histamine release test may further comprise ananti-histamine antibody and histamine as a standard.

In one embodiment, the present invention provides a method ofdiagnosing, or assisting diagnosis of, a sweat allergy or a diseaserelating to a sweat allergy antigen, comprising contacting

(i) the sweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide provided in the first orsecond aspect of the present invention, or

(ii) an antibody or an antibody fragment specifically binding to thesweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide provided in the fourthaspect of the present invention

with a sample derived from a human (a blood sample (e.g., plasma,serum), sweat, a sample derived from skin (e.g., skin washings)). Themethod may be performed in vitro.

In one embodiment, the present invention provides use of

(i) the sweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide provided in the first orsecond aspect of the present invention, or

(ii) an antibody or an antibody fragment specifically binding to thesweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide provided in the fourthaspect of the present invention

for manufacturing the composition for diagnosing, or assisting diagnosisof, a sweat allergy or a disease relating to a sweat allergy antigen.

8. Composition for Treating Sweat Allergy or Disease Relating to SweatAllergy Antigen

In an eighth aspect, the present invention provides a therapeuticcomposition for a sweat allergy or a disease relating to a sweat allergyantigen comprising a sweat allergy antigen protein that is a proteinderived from a microorganism or the MGL_1304 partial peptide.

The therapeutic composition for a sweat allergy or a disease relating toa sweat allergy antigen may be a composition for a hyposensitizationtherapy. The “hyposensitization therapy” is a method of treatmentperformed by administering a slight amount of a therapeutic allergen foran allergy associated with IgE antibody in a gradually increasing amountat intervals of a certain number of days so as to avoid occurrence of anallergic reaction even when a causative allergen enters. The protein orthe peptide provided in the first or second aspect of the presentinvention may be used as the therapeutic allergen in thehyposensitization therapy.

The therapeutic composition provided by the present invention isappropriately formulated using the protein or the peptide provided bythe present invention. For example, the therapeutic composition providedby the present invention can be formulated with pharmaceuticallyacceptable carriers (including additives). The pharmaceuticallyacceptable carriers include, but not limited to, excipients (e.g.,dextrin, hydroxypropyl cellulose, and polyvinylpyrrolidone),disintegrators (e.g., carboxymethyl cellulose), lubricants (e.g.,magnesium stearate), surfactants (e.g., sodium lauryl sulfate), solvents(e.g., water, saline, and soybean oil), and preservatives (e.g.,p-hydroxybenzoic acid ester).

A dosage and an administration method of the therapeutic composition mayappropriately be selected by those skilled in the art depending on anage, a body weight, and a health condition of a subject to beadministered.

In one embodiment, the present invention provides a method of treating asweat allergy or a disease relating to a sweat allergy antigencomprising administering the sweat allergy antigen protein that is aprotein derived from a microorganism or the MGL_1304 partial peptideprovided in the first or second aspect of the present invention.

In one embodiment, the present invention provides use of the sweatallergy antigen protein that is a protein derived from a microorganismor the MGL_1304 partial peptide provided in the first or second aspectof the present invention for manufacturing a therapeutic composition fora sweat allergy or a disease relating to a sweat allergy antigen.

9. Composition for Removing or Neutralizing Sweat Allergy Antigen andMaterial for Removing Sweat Allergy Antigen

In a ninth aspect, the present invention provides a composition forremoving or neutralizing a sweat allergy antigen and a material forremoving a sweat allergy antigen comprising a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide.

An antibody specifically binding to the sweat allergy antigen protein orthe MGL_1304 partial peptide can be used for neutralizing the antigenactivity of the sweat allergy antigen. The antibody can be also used forremoving the sweat allergy antigen from an affected part.

For example, an isotonic solution such as a saline comprising anantibody specifically binding to the sweat allergy antigen protein orthe MGL_1304 partial peptide may be contacted with the affected part toneutralize the sweat allergy antigen and/or to achieve removable of thesweat allergy antigen.

The antibody specifically binding to the sweat allergy antigen proteinor the MGL_1304 partial peptide may be immobilized to fibers and used asa material for removing the sweat allergy antigen from the affectedpart. An example of the material for removal is a wiping sheet. Thematerial for removal can appropriately be manufactured by those skilledin the art.

For example, Japanese Patent No. 3642340 discloses a method ofmanufacturing a material for removal such as a wiping sheet comprisingimmobilizing an antibody to fibers (woven fabric or nonwoven fabric)with an official moisture regain of 7% or more.

Examples of a method of immobilizing an antibody to a carrier such asfibers include a method in which after the carrier is silanized usingγ-aminopropyltriethoxysilane etc., an aldehyde group is introduced to acarrier surface by means of glutaraldehyde etc. to covalently bind thealdehyde group and the antibody; a method in which an untreated carrieris immersed in an aqueous solution of the antibody to immobilize theantibody to the carrier though ion binding; a method in which analdehyde group is introduced to a carrier having a certain functionalgroup to covalently bind the aldehyde group and the antibody; a methodin which the antibody is subjected to ion-binding to a carrier having acertain functional group; and a method in which after a carrier iscoated with a polymer having a certain functional group, an aldehydegroup is introduced to covalently bind the aldehyde group and theantibody. The certain functional group as described above may be an NHRgroup (R is an alkyl group of any of methyl, ethyl, propyl, and butylother than H), an NH₂ group, a C₆H₅NH₂ group, a CHO group, a COOH group,and an OH group. The antibody may be supported via a linker on thecarrier, and examples of the linker used include maleimide, NHS(N-Hydroxysuccinimidyl) ester, imidoester, EDC(1-Etyl-3-[3-dimetylaminopropyl]carbodiimido) and PMPI(N-[p-Maleimidophenyl]isocyanete).

The material for removal may be impregnated with water containingglycerol to be stored.

In one embodiment, the present invention provides a method of removingor neutralizing a sweat allergy antigen comprising contacting theantibody specifically binding to the sweat allergy antigen protein orthe MGL_1304 partial peptide provided in the fourth aspect of thepresent invention with the sweat allergy antigen.

In one embodiment, the present invention provides use of the antibodyspecifically binding to the sweat allergy antigen protein or theMGL_1304 partial peptide provided in the fourth aspect of the presentinvention for manufacturing a composition for removing or neutralizing asweat allergy antigen or a material for removing a sweat allergyantigen.

10. Composition or Kit for Determining Effect of HyposensitizationTherapy

In a tenth aspect, the present invention provides a composition or kitfor determining an effect of a hyposensitization therapy comprising asweat allergy antigen protein that is a protein derived from amicroorganism or the MGL_1304 partial peptide.

The hyposensitization therapy can be performed by administering acomposition comprising a sweat allergy antigen (e.g., the proteinprovided in the first aspect or the peptide provided in the secondaspect of the present invention) in a gradually increasing amount asdescribed in the eighth aspect of the present invention.

In general, it is known that the blood concentration of IgG4 against theantigen is increased while the blood concentration of IgE against theantigen is decreased in the course of treatment in the hyposensitizationtherapy.

Also in the hyposensitization therapy performed by administering acomposition comprising a sweat allergy antigen (e.g., the proteinprovided in the first aspect or the peptide provided in the secondaspect of the present invention) in a gradually increasing amount atintervals of a certain number of days, if the administration showstherapeutic effects, the blood concentration of IgG4 against the sweatallergy antigen may be increased, and the blood concentrations of IgEantibody and total IgG antibody against the sweat allergy antigen may bedecreased in the course of treatment.

Therefore, in one embodiment, the present invention provides a method ofdetermining an effect of a hyposensitization therapy comprisingmeasuring an amount of a human IgG4 antibody binding to a sweat allergyantigen (e.g., the protein provided in the first aspect or the peptideprovided in the second aspect of the present invention) in a human bloodsample (e.g., human serum or plasma). In one embodiment, the presentinvention provides a method of determining an effect of ahyposensitization therapy comprising comparing an amount of a human IgG4antibody binding to a sweat allergy antigen (e.g., the protein providedin the first aspect or the peptide provided in the second aspect of thepresent invention) in a blood sample collected before performing thehyposensitization therapy with a blood sample collected after performingthe hyposensitization therapy. In these methods of determining an effectof a hyposensitization therapy, if the concentration of IgG4 antibodybinding to a sweat allergy antigen in blood of a patient increases, itcan be determined that the hyposensitization therapy exerts an effect.The methods of determining an effect of a hyposensitization therapy mayfurther comprise measuring the amounts of IgE antibody and/or total IgGantibody in the blood to the sweat allergy antigens, and/or comparingthe amounts of the IgE antibody and/or total IgG antibody binding to thesweat allergy antigen in a blood sample collected before performing thehyposensitization therapy with a blood sample collected after performingthe hyposensitization therapy. The methods of determining an effect of ahyposensitization therapy may be performed in vitro.

The composition or kit for detecting or quantifying an antibody bindingto a sweat allergy antigen provided in the sixth aspect of the presentinvention can be utilized in the measurement of an amount of an antibodybinding to the sweat allergy.

Therefore, the composition or kit comprising a sweat allergy antigenprotein that is a protein derived from a microorganism or the MGL_1304partial peptide provided in the sixth aspect of the present inventioncan be used as a composition or kit for determining an effect of ahyposensitization therapy.

In one embodiment, the present invention provides use of a sweat allergyantigen protein that is a protein derived from a microorganism providedin the first aspect of the present invention or the MGL_1304 partialpeptide provided in the second aspect of the present invention formanufacturing a composition or kit for determining an effect of ahyposensitization therapy.

The present invention will hereinafter further be described in examplesand these examples are not intended to limit the present invention.

EXAMPLES Example 1 Purification of Partially Purified Sweat Antigen(QRX)

1-1. Preparation of Concentrated Sweat

After insolubles were removed from a human sweat through 100-μm and70-μm mesh filters (Nylon Cell Strainers, Falcon), precipitates werefurther removed by a 0.22-μm filter (Bottle Top Filter, 1 L, Corning).Four litters of the sweat filtrated by the filters were concentrated byultrafiltration (3000M.W.cut) to about 150 mL and used as a material forthe sweat antigen purification.

1-2. Separation by Means of Anion-Exchange Column

To an anion-exchange column MonoQ 10/100 GT (GE Healthcare Bio-Sciences)preliminarily equilibrated by 10 mmol/L Tris-HCl (pH 8.0), 75 mL of theconcentrated sweat prepared at pH 8.0 was loaded, and was eluted through0 to 1.0 M NaCl concentration gradient in 10 mmol/L Tris-HCl (pH 8.0).AKTA Explorer (GE Healthcare Bio-Sciences) was used as a chromatographicdevice for purification.

To select a fraction containing a substance inducing the histaminerelease activity, a histamine release test using basophils of an atopicdermatitis patient was performed for each of fractions.

First, each of the appropriately diluted fractions was mixed at 1:1 witha basophil fraction of an atopic dermatitis patient prepared in a HEPESbuffer containing 5 mmol/L glucose, 0.03 w/v % HSA, 2 mmol/L CaCl₂, and1 mmol/L MgCl₂, and was incubated at 37° C. for 40 minutes. Aftersupernatant and sedimented blood cells were separated by centrifugationand were respectively denatured by adding 0.2 mol/L perchloric acid, ahistamine concentration in the supernatant obtained by thecentrifugation was measured by HPLC (Shimadzu LC solution). A rate ofthe histamine amount of the supernatant relative to the total histamineamount was defined as the histamine release activity.

The histamine amount was measured in accordance with a method describedin a literature (Koro, O. et al., J. Allergy Clin. Immunol., 103,663-670, 1999).

As a result, a fraction eluted within a salt concentration range of 0.25to 0.3 mol/L NaCl having the histamine release activity higher than theother fractions was recovered as a fraction exhibiting the histaminerelease activity.

1-3. Separation by Means of Reverse-Phase Column

After diluting 18 mL of the fraction acquired in Example 1-2 with purewater 10 times, TFA was added at a final concentration of 0.1 v/v %.This was loaded to a reverse-phase column (SOURCE 15RPC ST 4.6/100 (GEHealthcare Bio-Sciences)) and was eluted through concentration gradientfrom 0.1 v/v % TFA/distilled water to 0.1 v/v % TFA/acetonitrile. AKTAExplorer (GE Healthcare Bio-Sciences) was used as a chromatographicdevice for purification.

After volatilizing TFA and acetonitrile of the eluted fractions, thehistamine release test was performed as in Example 1-2.

As a result, a fraction within a range of about 30 to 35 v/v %acetonitrile having the histamine release activity higher than the otherfractions was recovered as the fraction exhibiting the histamine releaseactivity (4 mL).

1-4. Separation by Means of Gel Filtration Chromatography

After lyophilization, the fraction obtained in Example 1-3 wasre-dissolved in PBS, loaded to Superdex 75 PC 3.2/30 (GE HealthcareBio-Sciences), and fractionated and eluted in PBS (−). Smart System (GEHealthcare Bio-Sciences) was used as a chromatographic device forpurification.

The histamine release test was performed for the eluted fractions as inExample 1-2.

As a result, a fraction within a range of elution positions from 15 to60 kD was recovered as the fraction exhibiting the histamine releaseactivity (1.2 mL) and subsequently defined as a QRX fraction.

Example 2 Purification and Mass Spectrometry of Partially Purified SweatAntigen (QRX)

The partially purified sweat antigen (QRX) was purified by using an Aqua5μ-C18-200A HPLC column (manufactured by Phenomenex) (eluted throughconcentration gradient from 0.1 v/v % TFA/distilled water to 0.1 v/v %TFA/100% acetonitrile). The fraction exhibiting the histamine releaseactivity was recovered and further purified by a Jupiter 5μ-C18-300AHPLC column (manufactured by Phenomenex) (eluted through concentrationgradient from 0.1 v/v % TFA/distilled water to 0.1 v/v % TFA/80%acetonitrile), and a mass spectrometry (TOF-MS) was performed for thefraction exhibiting the histamine release activity (fraction of an arrowof FIG. 1). Although a sample is normally cationized in TOF-MS, the massmeasurement was performed through anionization in this experiment. Thehistamine release activity was measured in accordance with a methoddescribed in Koro, O. et al., J. Allergy Clin. Immunol., 103, 663-670,1999. The detected amino acid sequence matched MGL_1304.

Example 3 Preparation of Recombinant Protein of MGL_1304 and Reactivityto Atopic Dermatitis Patient IgE

Malassezia globosa was purchased from ATCC (MYA-4612). Reversetranscription of mRNA extracted from Malassezia globosa to cDNA wasperformed and the cDNA encoding MGL_1304 was amplified by PCR (senseprimer: 5′-GGGGTACCGTATCCCTCAACATTTTCTCAGCTGC-3′ (SEQ ID NO: 8);antisense primer: 5′-CCCAAGCTTTTAGCAGTCGTACTTGCCGGGGATG-3′ (SEQ ID NO:9), (94° C., 5 min/60° C., 1 min/72° C., 1 min)×1 cycle, (94° C., 1min/60° C., 1 min/72° C., 1 min)×30 cycles, (94° C., 5 min/60° C., 1min/72° C., 10 min)×1 cycle) and the amplified cDNA was inserted intothe p Cold TF vectors (manufactured by TAKARA BIO INC.) followed bytransfection of E. coli JM109 with the obtained vector. After culturingat 15° C. for 24 hours, the resulting E. coli was dissolved in xTractorbuffer and a recombinant protein was purified by a cobalt column. Aprotein with Trigger Factor only (TF), a TF-MGL_1304 fusion protein(TF-MGL_1304), and a protein obtained by removing TF from the fusionprotein through enzyme treatment (rMGL_1304) were prepared. The obtainedproteins were subjected to acrylamide gel electrophoresis and weredirectly CBB-stained (left side of FIG. 2) or transferred to PVDFmembranes for immunoblotting with an anti-His tag antibody (center ofFIG. 2) and an atopic dermatitis patient serum (right side of FIG. 2).The results indicate that the atopic dermatitis patient IgE binds torMGL_1304 (FIG. 2).

The prepared rMGL_1304 was reacted with atopic dermatitis patientperipheral blood basophils (FIG. 3, AD1, AD2, and AD3) and healthyperson peripheral blood basophils (FIG. 3, HC1) to perform the histaminerelease test. The results indicated that MGL_1304 induces histaminerelease specifically in the atopic dermatitis patients.

Additionally, cDNA encoding the MGL_1304 described above or mite cDNA isinserted into pSecTag2/Hygro vector (manufactured by Invitrogen)containing Myc-tag to transfect COS7 cells. A culture supernatant of theCOS7 cells transfected with these DNAs was subjected to acrylamide gelelectrophoresis and transferred to a PVDF membrane for immunoblottingwith anti-Myc tag antibodies. The results indicated that the culturesupernatant contains proteins corresponding to respective cDNAs (In FIG.11, A). The culture supernatant was further reacted with atopicdermatitis patient peripheral blood basophils to perform the histaminerelease test. The same basophils were reacted with sweat antigens (QR)partially purified from the concentrated human sweat by theanion-exchange column chromatography and the reverse-phase columnchromatography to perform the histamine release test for comparison withthe histamine release rate from the culture supernatant (In FIG. 11, B).

The results indicated that MGL_1304 protein (rMGL_1304) produced by theCOST cells induces histamine release like the partially purified humansweat antigens (QR).

Example 4 Reactivity Between Recombinant Protein of MGL_1304 and AtopicDermatitis Patient IgE

Experiments were conducted to study whether MGL_1304 had substantiallythe same property as the partially purified sweat antigen (QRX) used sofar.

(1)

Recombinant mite antigens (Der f1), QRX, and MGL_1304 wereelectrophoresed and transferred to PVDF membranes to prepare a pluralityof membranes. An atopic patient serum (AD serum) pretreated with QRX orMGL_1304 prepared in Example 3 or AD serum without pretreatment was usedfor immunoblotting. The atopic dermatitis patient serum used wasobtained from three patients. The pretreatment with MGL_1304 inhibitedthe binding of IgE to QRX and the pretreatment with QRX inhibited thebinding of IgE to MGL_1304 (FIG. 4).

(2)

The atopic dermatitis patient serums (AD1 to AD4) were pretreated withTF or TF-MGL_1304 and used for sensitizing a rat cell line expressinghuman IGE receptors (sub-unit A) so as to measure degranulation whenbeing stimulated with anti-IgE, QRX, and a mite extract (Mite-Df). Theresults indicated that when MGL_1304 specific IgE is removed bypretreatment with MGL_1304 prepared in Example 3, the reactivity to QRXstimulation disappears (FIG. 5).

Example 5 Structure Necessary for IgE Binding in MGL_1304

MGL_1304 proteins (183 amino acids) truncated at the N-terminal,truncated at the C-terminal, and corresponding to 1-50 (P1), 46-100(P2), 96-140 (P3), and 146-183 (P4) of the polypeptide represented bySEQ ID NO:1 were expressed in E. coli to be prepared, and immunoblottedwith anti-His tag antibodies, atopic dermatitis patient serums (In FIG.6, B; AD1, AD2, AD3, and AD4), and/or the Smith2 antibodies, which areantibodies produced by the hybridoma (Mouse-Mouse hybridoma smith-2) ofAccession No. FERM BP-11111 (transferred from FERM P-21440) deposited tothe International Patent Organism Depositary of the National Instituteof Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki 305-8566) on Apr. 1, 2009. For peptide fragments, thehistamine release test was performed using the atopic dermatitis patientbasophils (In FIGS. 6, C and D). Numbers denote the numbers of the aminoacid sequences.

The results indicated that the protein with the N-terminal shortened by32 or more amino acids or with the C-terminal deleted by 10 or moreamino acids loses the binding ability to the atopic dermatitis patientIgE. Neither the binding ability to the atopic dermatitis patient IgEnor the histamine release activity was shown by polypeptides P1 to P4prepared by fragmenting MGL_1304 polypeptide into four pieces having ashort overlap with each other (P1: a polypeptide corresponding to theamino acid sequence 1-50 of the MGL_1304 protein (SEQ ID NO: 10); P2: apolypeptide corresponding to the amino acid sequence 46-100 of theMGL_1304 protein (SEQ ID NO: 11); P3: a polypeptide corresponding to theamino acid sequence 96-140 of the MGL_1304 protein (SEQ ID NO: 12); P4:a polypeptide corresponding to the amino acid sequence 136-183 of theMGL_1304 protein (SEQ ID NO: 13). On the other hand, the Smith-2antibody exhibits the binding property to both the MGL_1304 protein andthe polypeptide (P1) corresponding to the amino acid sequence 1-50thereof (In FIG. 6, E). These results indicated that the atopicdermatitis patient IgE has a high possibility of recognizing ahigher-order structure rather than a short peptide constituting theMGL_1304 protein (In FIG. 6, A). The results also indicated that the P1(MGL_1304 protein and the polypeptide corresponding to the amino acidsequence 1-50 thereof) region may be a recognition site of ananti-MGL_1304 protein antibody in the MGL_1304 protein.

Example 6 ELISA Using MGL_1304

rMGL_1304 prepared in Example 3 was coated onto a 96-well microplate,followed by blocking using bovine serum albumin (BSA), and subsequently,serums of 4 AD patients (including AD2 who is HRT-negative) and ahealthy volunteer (Normal) were added thereto. The results of detectingIgE binding to rMGL_1304 with the anti-human IgE antibody are shown(FIG. 7). It was revealed that use of a recombinant MGL_1304 proteinallows measurements of MGL_1304-specific IgE in serum (FIG. 7).

Example 7 Immunoreactivity of Malassezia Fungus Body (M. globosa)Extract, Malassezia Fungus Body Culture Supernatant, QRX, andRecombinant MGL_1304 Protein

M. Globosa was cultured in a 2693 mDixon medium at 32° C. for four days.The culture medium was centrifuged (2000 rpm) to separate a culturesupernatant and a fungus body. The fungus body was dissolved in PBS,fragmented with ultrasonic waves, and centrifuged (2000 rpm), and thesupernatant was recovered to prepare a fungus body extract through a0.22-μm filter. The Malassezia fungus body extract, the medium only, theMalassezia culture supernatant, the MGL_1304 protein (rMGL) prepared inExample 3, and QRX are electrophoresed and immunoblotted with eachatopic dermatitis patient serum from two patients (in FIGS. 8 and 9, theatopic dermatitis patient serums used are different).

After purification of the Malassezia fungus body extract and theMalassezia culture supernatant, the purified Malassezia fungus bodyextract, the purified Malassezia culture supernatant, and QRX wereelectrophoresed and immunoblotted with the Smith2 antibody (FIG. 10).The Malassezia fungus body extract and the Malassezia culturesupernatant were purified as follows. M. Globosa was cultured in a 2693mDixon medium at 32° C. for four days. The culture medium wascentrifuged (2000 rpm) and the supernatant was recovered to prepare theMalassezia culture supernatant through a 0.22-μm filter. The culturesupernatant was fractionated by the ion-exchange column chromatographyand the reverse-phase column chromatography described in Example 1 usingthe histamine release as an index, so as to recover a fraction havingthe histamine release activity.

Although rMGL exhibited a molecular weight (about 23 kDa) expected fromthe DNA sequence (FIG. 2), a protein detected in the atopic dermatitispatient serum, which is present in the lysate of the Malassezia fungusbody, exhibited about 30 k Da, indicating that MGL is subjected topost-translational modification (FIGS. 8, 9, and 10). On the other hand,a protein detected in the atopic dermatitis patient serum, which ispresent in the culture supernatant of the Malassezia fungus body,exhibited about 17 kDa, which is the same as QRX (FIG. 10). Therefore,it was revealed that MGL is further modified when it is secreted fromthe fungus body. A band of about 30 kDa present in the lysate of theMalassezia fungus body, a band of about 17 kDa present in the cultureserum of the Malassezia fungus body, and a band of QRX disappeared whenthe atopic dermatitis patient serum was pretreated with rMGL (FIGS. 8and 9). In terms of the histamine release activity, the purified antigenfrom the culture supernatant secreted from the Malassezia fungus bodyshows higher activity compared to rMGL (full-length rMGL obtained by anenzyme treatment to cleave TF bound to rMGL for the protein expression).

Example 8 Binding of Antibody Raised Against Recombinant MGL_1304Protein as Immunogen to QRX and Recombinant MGL_1304 Protein

The recombinant MGL_1304 protein (rMGL_1304) prepared in Example 3 wasused for immunization of Balb/c mice, and screening was performed byusing the binding property to rMGL as an index so as to prepare amonoclonal antibody producing strain (MGLab). The binding properties ofthese antibodies to the polypeptides (P1 to 4) prepared in Example 5having the sequences obtained by fragmenting the MGL_1304 protein intofour pieces were examined by ELISA.

P1 to P4 fused with TF (TF-P1 to 4) dissolved in PBS resulting in 3μg/ml solution were coated on a 96-well microplate (50 μl/well) andblocked by BSA, and the monoclonal antibodies were added to detect mouseIgG binding to the polypeptides using enzyme-labeled anti-mouse IgGantibodies.

As a result, MGLab6-3 bound to P4 while MGLab8-2, 8-4, 9-1, 9-5, 10-8,10-10, 22-1, 36-1, and 40-1 bound to P2, indicating preparation ofmonoclonal antibodies which recognize epitopes different from Smith-2described in Example 3 (FIG. 12).

The results indicated that the P2 (the polypeptide corresponding to theamino acid sequence 46-100 (Seq ID NO: 11) of the MGL_1304 protein) andP4 (P4: the polypeptide corresponding to the amino acid sequence 136-183(Seq ID NO: 13) of the MGL_1304 protein) regions may be recognitionsites of an anti-MGL_1304 protein antibody in the MGL_1304 protein.

Example 9 Measurement of Anti-Sweat Antigen-Specific IgE Antibody inPatient Serum

Monoclonal antibodies MGLab8-2 (8-2) and MGLab6-3 (6-3) prepared byimmunizing mice with rMGL prepared in Example 3 were used for studyingwhether IgE antibodies in serums of atopic dermatitis patients (AD1 andAD2) can be detected.

A 96-well ELISA plate was coated with the antibodies at 10 μg/ml and 50μl/well and was left overnight at 4° C. The plate was washed twice,blocked by 2% BSA (one hour), and then washed twice. After 100-folddiluted QRX or 3 μg/ml of TF-MGL prepared in Example 3 was added at 100μl/well, left for 90 minutes, and washed three times, the serum wasadded at 100 μl/well and left for 90 minutes. After the plate was washedthree times, a solution containing HRP-labeled anti-human IgE antibodyis added at 100 μl/well, left for one hour, and washed three times, anda color was developed by using TMB to measure absorbance (450 nm).

As a result, the monoclonal antibody MGLab8-2 prepared by immunizingmice with rMGL detected the IgE antibody in the serums of the atopicdermatitis patients via binding to QRX or rMGL. On the other hand, theSmith2 antibody was not able to detect the IgE antibody in the serums ofthe atopic dermatitis patients via rMGL (FIGS. 13 to 15).

Then, experiments were conducted to study whether the IgE antibody inthe serum of the atopic dermatitis patient can be quantified by means ofthe monoclonal antibodies MGLab8-2 and MGLab6-3 prepared by immunizingmice with rMGL. As a result, the absorbance was decreased in proportionto a decrease in an IgE antibody amount due to dilution of the serum ofthe atopic dermatitis patient. It was revealed that the both antibodies(monoclonal antibodies MGLab8-2 and MGLab6-3) enable quantification ofthe IgE antibodies in the serum of the atopic dermatitis patient usingrMGL (FIGS. 16 and 17).

These results indicated that a sweat allergy in atopic dermatitis can bediagnosed by utilizing the monoclonal antibody prepared by immunizingmice with rMGL.

Example 10 Measurement of Anti-Sweat Antigen-Specific IgE Antibody inPatient Serum (2)

Experiments were conducted to study whether IgE antibody specificallybinding to a sweat antigen can be detected in serums of atopicdermatitis (AD), allergic rhinitis, and a healthy person (Normal) byusing ELISA coating rTF-MGL prepared in Example 3.

A 96-well ELISA plate was coated with rTF or rTF-MGL at 3 μg/ml and 50μl/well and was left overnight at 4° C. The plate was washed twice,blocked by 2% BSA (room temperature, one hour), and then washed twice.The serum diluted by 1% BSA was added at 100 μl/well and left for onehour at room temperature. After the plate was washed three times, asolution containing HRP-labeled anti-human IgE antibodies is added at100 μl/well, left for one hour, and washed three times, and a color wasdeveloped by using TMB to measure absorbance (450 nm).

As a result, IgE binding to the sweat antigen (MGL) was detected in theserums of the atopic dermatitis patients and the allergic rhinitispatients by ELISA coating rMGL (FIGS. 18 and 19). On the other hand, IgEbinding to the sweat antigen (MGL) was not detected in the serum of thehealthy person (FIGS. 18 and 19).

These results indicated that a sweat allergy can be diagnosed in atopicdermatitis and allergic rhinitis patients by ELISA coating rMGL.

Example 11 Measurement of Anti-MGL Antibody in Patient Subjected toHyposensitization Therapy

QRX was repeatedly subcutaneously injected to a sweat allergy patient atconcentration gradually increased from a low concentration. Serums werecollected over time and changes in the anti-MGL antibody amount in thecollected serums were measured by ELISA using the anti-human IgEantibody, the anti-human IgG antibody, or the anti-human IgG4 antibody,where rMGL prepared in Example 3 was coated.

As a result, changes in respective antibody amounts of the anti-MGL-IgEantibody, the anti-MGL-IgG antibody, and the anti-MGL-IgG4 antibody weremeasured in patient serums (FIGS. 20 to 22). Although the concentrationsof the anti-MGL-IgE and the anti-MGL-IgG (including sub-types 1 to 4)was decreased in the course of treatment, the anti-MGL-IgG4 wasincreased in the course of treatment. This phenomenon is consistent witha general knowledge that IgE is slightly decreased while theconcentration of IgG4 against the antigen thereof at first rises in thehyposensitization therapy.

According to a clinical diagnosis by a physician, DLQI, which is anindex indicative of quality of life, was improved by due to thehyposensitization therapy applied to the patient.

These results indicated that rMGL is useful as an antigen for thehyposensitization therapy as is the case with QRX, and also indicatedthat rMGL is useful for determination of a therapeutic effect of thehyposensitization therapy.

Example 12 Study on Production Amount of MGL_1304 Protein in CulturingUsing Buffer Solution of M. globosa

M. Globosa was cultured in a 2693 mDixon medium at 32° C. for four days.The culture supernatant was removed and the fungus body was washed witha buffer solution (buffer A: PBS/HEPES/glucose buffer solution) at pH 4,pH 6, or pH 8. The buffer solutions were subsequently used in culturingfor 2 to 60 minutes and a concentration of the produced MGL_1304 proteinwas measured by ELISA. As a result, the production of the MGL_1304protein was increased when the culture was performed using the buffersolution at pH 8 (FIG. 23).

The invention claimed is:
 1. A method of detecting antibody binding to asweat allergy antigen protein, comprising contacting a biological samplefrom a human with a sweat allergy antigen protein selected from thegroup consisting of (i) a protein comprising an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 1; and (ii) a peptide comprising an amino acid sequence having90% or more sequence identity to the amino acid sequence of any of SEQID NO: 2 to 7; and detecting the antibody binding to the sweat allergyantigen protein.
 2. The method of claim 1, further comprising measuringan amount of the antibody binding to a sweat allergy antigen protein. 3.The method of claim 1, further comprising determining an amount of theantibody binding to a sweat allergy antigen protein in the sample basedon results from detecting the antibody that binds to the protein orpeptide, wherein the contacting the sample with the sweat allergyantigen protein is performed using the sweat allergy antigen protein ata plurality of known concentrations.
 4. The method of claim 1, whereinthe sweat allergy antigen protein is a protein comprising the amino acidsequence of SEQ ID NO:
 1. 5. The method of claim 1, wherein the sweatallergy antigen protein is a protein consisting of the amino acidsequence of SEQ ID NO:
 1. 6. The method of claim 1, wherein the sweatallergy antigen protein is a peptide comprising the amino acid sequenceof any of SEQ ID NO: 2 to
 7. 7. The method of claim 1, wherein the sweatallergy antigen protein is a peptide consisting of the amino acidsequence of any of SEQ ID NO: 2 to
 7. 8. The method of claim 1, whereinthe biological sample from a human includes sweat, skin washings, asolution of an extract of skin, a serum, or a plasma.
 9. The method ofclaim 1, wherein the detecting the antibody is performed using Westernblotting or enzyme-linked immunosorbent assay (ELISA).
 10. The method ofclaim 1, wherein the sweat allergy antigen protein is a protein secretedfrom a fungus body of Malassezia globosa, has a molecular weight ofabout 17 kDa, and binds to (i) serum derived from a sweat allergypatient and/or (ii) smith2 antibody.
 11. The method of claim 1, whereinthe sweat allergy antigen protein is a protein present in a fungus bodyof Malassezia globosa, has a molecular weight of about 30 kDa, and bindsto (i) serum derived from a sweat allergy patient and/or (ii) smith2antibody.
 12. A method of diagnosing a sweat allergy or a diseaserelating to a sweat allergy antigen protein, comprising detectingantibody binding to a sweat allergy antigen protein in a sample from ahuman according to the method of claim 1.